CagA mediates epigenetic regulation to attenuate let-7 expression in Helicobacter pylori-related carcinogenesis.

OBJECTIVE: MicroRNAs (miRNAs) act as tumour suppressor genes or oncogenes in the regulation of multiple carcinogenic processes. Aberrant miRNA expression is reported in Helicobacter pylori (H pylori)-related gastritis and gastric cancer. The cytotoxin-associated gene A (CagA) of H pylori has a pathophysiologically important role in gastric carcinogenesis. A study was undertaken to evaluate the effect of CagA on miRNA expression and its regulatory mechanism. METHODS: The effect of CagA on miRNA expression was assessed by comprehensive miRNA microarray. The mechanisms of the in vitro and in vivo effects of CagA on histone modification and DNA methylation and the involvement of CagA-dysregulated signal transduction on let-7, an important representative miRNA in gastric carcinogenesis, were investigated. RESULTS: In in vitro experiments, CagA significantly attenuated let-7 expression leading to Ras pathway activation. CagA enhanced c-myc, DNA methyltransferase 3B (DNMT3B) and Enhancer of Zeste homologue 2 (EZH2) expression and attenuated miR-26a and miR-101 expression, which resulted in the attenuation of let-7 expression by histone and DNA methylation. Experiments performed in CagA transgenic mice revealed that c-myc, EZH2 and DNMT3B expression were enhanced and let-7 expression was attenuated to induce Ras oncoprotein expression in the stomach, with no associated inflammation. CONCLUSIONS: H pylori CagA induces aberrant epigenetic silencing of let-7 expression, leading to Ras upregulation.

A case of collagenous gastritis resembling nodular gastritis in endoscopic appearance.

A 25-year-old Japanese female was referred to our clinic for the investigation of moderate iron-deficiency anemia and epigastralgia. Endoscopic examination showed diffuse mucosal nodules in the gastric body resembling nodular gastritis, but this pattern was not observed in the antrum. Histology of the gastric biopsies taken from the gastric body showed mild atrophic mucosa with chronic active inflammation. Some of the biopsy specimens showed deposition of patchy, band-like subepithelial collagen. Four years later, the patient showed no clinical symptoms and signs. A follow-up endoscopic examination showed similar findings, which mimicked pseudopolyposis or a cobblestone-like appearance. The biopsy specimens from the depressed mucosa between the nodules revealed a thickened subepithelial collagen band with no improvement, which led to a diagnosis of collagenous gastritis. Treatment with oral administration of proton-pump inhibitors and histamine-2-receptor antagonists had proved ineffective. To make a correct diagnosis of collagenous gastritis, we should determine the characteristic endoscopic findings and take biopsies from the depressed mucosa between the nodules.

Restoration of gut motility in Kit-deficient mice by bone marrow transplantation.

PURPOSE: Interstitial cells of Cajal (ICC) play important roles in autonomic gut motility as electrical pacemakers and mediators of neural regulation of smooth muscle functions. Insufficiency of ICC has been reported in a wide range of gut dysmotilities. Thus, restoration of ICC may be a therapeutic modality in these diseases. Here we provide evidence that transplanted bone marrow (BM) cells can restore gut dysmotility in part via transdifferentiation to ICC. METHODS: Bone marrow cells obtained from Kit insufficient W/W(v) mice or syngeneic GFP-transgenic mice with wild-type Kit were transferred to W/W(v) recipients. Whole gut transit time and gastric emptying were examined 5 and 6 weeks after BM transplantation, respectively, and ICCs were identified in whole mounts, frozen sections and transmission electron immunomicroscopy of the gut smooth muscle layers using specific antibodies. RESULTS: Transplantation of wild-type BM into W/W(v) mice significantly improved whole gut transit time and gastric emptying. Fluorescent immunohistochemistry revealed GFP(+)Kit(+) cells in the myenteric plexus, deep muscular plexus, and submucosal plexus smooth muscle layers of the stomach, small intestine, and colon, respectively. In the whole mounts, GFP(+)Kit(+) cells were bipolar and spindle shaped, and transmission electron immunomicroscopy showed GFP(+) cells rich in mitochondria and endoplasmic reticulum between gut smooth muscle layers, suggesting the presence of GFP(+) cells with morphological characteristics of ICC. CONCLUSIONS: These results suggest that BM contains cells that may incorporate into ICC networks and improve dysmotility in W/W(v) mice. Thus, BM transplantation may become to a new therapeutic modality for gut dysmotilities due to ICC insufficiency.

Topical implantation of mesenchymal stem cells has beneficial effects on healing of experimental colitis in rats.

Mesenchymal stem cells (MSCs) are attractive cell sources in regenerative medicine. We examined the effects of topical MSCs implantation on an experimental model of inflammatory bowel disease. Putative MSCs, isolated from bone marrow aspirates of male rats by dish adherence and expanded in vitro, were characterized by flow cytometry, reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and differentiation assays. Experimental colitis was induced by intraluminal instillation of 2,4,6-trinitrobonzene sulfonic acid (TNBS) in the colons of male rats. The putative MSCs and unselected fresh bone marrow cells were injected into the colonic submucosa surrounding the area exposed to TNBS. The healing process of the injury was examined macroscopically and immunohistologically. Multipotent MSCs positive for CD29 and CD90, and negative for CD31 and CD34, were implanted into colon tissue surrounding the lesion; a majority of the engrafted cells were positive for vimentin. The implantation significantly accelerated healing of the damaged mucosa compared with vehicle-injected controls. The MSCs expressed vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-beta1 in vitro and after the implantation. In conclusion, we found that MSCs were successfully topically implanted in the colon and that they were associated with accelerated healing of TNBS-induced colitis. The beneficial effects of the MSCs might be mediated, at least in part, by their ability to differentiate into colonic interstitial cells and by their ability to provide VEGF and TGF-beta1 to the injured area.

Cultured bone marrow cell local implantation accelerates healing of ulcers in mice.

BACKGROUND: The therapeutic potential of bone marrow (BM)-derived cells in ulcers is not known. This study aimed to clarify (1) cell types that are derived from the BM which infiltrate ulcers; (2) whether BM-derived cells or gastric myofibroblasts can be used for cell transplantation to treat ulcers; and (3) the phenotypes of such transplantable cells. METHODS: (1) Wild-type mice were transplanted with BM cells of green fluorescent protein (GFP)-transgenic mice. Acetic acid-induced gastric ulcers were produced in mice after BM transplantation. (2) BM cells and gastric myofibroblasts were isolated from GFP-transgenic mice. Bone marrow cells attached to plastic dishes were selected for expansion. Gastric ulcers were induced, and BM-derived cells, myofibroblasts, or phosphate-buffered saline were injected around ulcers. The ulcer healing process was examined macroscopically and histologically. (3) Expression of growth factors and cytokines in transplantable cells was examined by reverse transcriptase-polymerase chain reaction. RESULTS: (1) GFP-positive cells with interstitial phenotypes were observed at the ulcerated area. (2) Ulcer healing was significantly promoted by the injection of BM-derived cells compared to controls on day 7, but not on day 3. The BM-derived cells were observed in the tissue surrounding the ulcer. However, myofibroblasts were not found. (3) The BM-derived cells expressed hepatocyte growth factor, transforming growth factor-beta(1), and other stromal factors before transplantation, and had mesenchymal phenotypes after transplantation. CONCLUSIONS: BM-derived cells are involved in the ulcer healing. BM-derived cells, but not myofibroblasts, are locally implantable to ulcers. Thus, BM-derived cells can be transplanted to accelerate ulcer healing.

Topical transplantation of mesenchymal stem cells accelerates gastric ulcer healing in rats.

Mesenchymal stem cells (MSCs), a subpopulation of adult somatic stem cells, are an attractive stem cell source in regenerative medicine because of their multipotentiality. We examined the effects of MSC transplantation on gastric ulcer healing. Putative MSCs, isolated from bone marrow aspirates of male rats by dish adherence and expanded in culture, were characterized by flow cytometry and reverse transcription-polymerase chain reaction. Gastric ulcers were induced by serosal application of acetic acid on the anterior wall of the stomach in female rats. Either MSCs (labeled with PKH67; 1x10(7) cells) or vehicle was injected into the gastric wall surrounding the ulcer. The healing process of the ulcer and the influence of anti-vascular endothelial growth factor (VEGF) antibody were examined. CD29-positive, CD90-positive, CD34-negative, and CD45-negative MSCs expressed mRNAs for VEGF and hepatocyte growth factor (HGF). The MSCs were transplantable to the gastric tissue surrounding the ulcer, where a majority of the engrafted cells were positive for vimentin. The transplantation significantly accelerated gastric ulcer healing compared with controls. The engrafted MSCs also expressed VEGF and HGF. Administration of anti-VEGF neutralizing antibody dose dependently reduced the MSC-induced promotion of ulcer healing. In conclusion, MSC transplantation accelerated gastric ulcer healing, possibly through the induction of angiogenesis in the gastric mucosa via the secretion of VEGF. The beneficial effects of MSCs might be mediated not only by their differentiation into gastric interstitial cells, but also by their ability to supply angiogenic factors.

Involvement of bone marrow-derived stromal cells in gastrointestinal cancer development and metastasis.

BACKGROUND: The involvement of bone marrow (BM) in tumor-stroma reactions or tumor development has not been examined in a cancer allograft, which has otherwise been appropriate for assessing therapeutic modalities. We investigated the fate of BM-derived cells in colon cancer allografts and liver metastases in mice. METHODS: C57BL/6 mice were irradiated and rescued by BM transplantation from green fluorescent protein (GFP)-transgenic mice. MC38 colon cancer cells were stably transfected with the pDsRed gene in order to identify tumor cells by fluorescence. These were inoculated into the mice to generate subcutaneous allografted tumors or liver metastases. The tumors were observed under confocal microscopy and fluorescent immunohistochemistry to determine the fate of tumor versus BM-derived cells. RESULTS: GFP-positive (GFP(+)) cells were consistently identified as vimentin(+), alpha-smooth muscle actin (alphaSMA)(+), spindle-shaped stromal cells in both the subcutaneous tumors and the liver metastases. GFP(+) cells of leukocyte lineage also infiltrated the tumors. Neither GFP(+) CD31(+) endothelial cells nor GFP(+) DsRed(+) cells were detected in the tumor. CONCLUSIONS: BM-derived cells frequently and consistently infiltrated the tumor allografts and metastases as interstitial cells and leukocytes. Cells derived from the fusion of BM cells and tumor cells were not observed. This model may be appropriate for the clarification of the effects of anticancer therapies and the study of BM-derived cells in tumor-host interactions.

Helicobacter pylori infection induces candidate stem cell marker Musashi-1 in the human gastric epithelium.

Musashi-1 (Msi-1), a mammalian neural RNA-binding protein, has been found to play important roles in the maintenance of stem cell states and differentiation in neural stem cells and mouse intestinal cells. We explored Msi-1 expression and its potential implications in the human stomach. Reverse transcription-PCR revealed that Msi-1 levels were significantly higher in the corpus than in antrum in Helicobacter pylori (Hp)-infected patients (n = 49) (P < 0.00001) in paired biopsy samples, whereas they were low and comparable at these two sites in Hp-negative patients (n = 31). Msi-1 levels were significantly higher in the Hp-infected corpus (n = 107) than in the Hp-negative corpus (n = 69) (P < 0.00000001). Immunohistochemistry and in situ hybridization demonstrated that Msi-1 was expressed at the base and neck/isthmus region of the fundic glands and partly co-expressed in Ki-67-positive cells in the corpus and antrum. Msi-1 levels correlated with Hp density (P < 0.05). Based on these results, we conclude that Hp infection strongly induces Msi-1 in the corpus. Given its expression in dividing cells, Msi-1 may modulate the state of gastric progenitor cells.

The transdifferentiation of bone-marrow-derived cells in colonic mucosal regeneration after dextran-sulfate-sodium-induced colitis in mice.

Bone-marrow-derived cells (BMDCs) transdifferentiate into various types of gastrointestinal cells. The precise transdifferentiation of BMDCs in gut regeneration, however, is controversial. In this study, we examined the transdifferentiation of BMDCs in the regeneration of damaged colonic epithelia. Lethally irradiated wild-type female mice (C57BL/6) were rescued by bone marrow transplantation from male green fluorescent protein transgenic mouse donors. Chronic colitis was induced by administering 3% dextran sulfate sodium (DSS) in the drinking water for 5 days on day 28 after the bone marrow transplantation. The mice were killed on day 25 after DSS administration. BMDC phenotypes were examined by confocal microscopy and fluorescence immunohistochemistry. BMDCs were frequently observed in the vimentin-positive colonic interstitial cells, which also expressed alpha-smooth muscle actin and had a spindle-like morphology, but did not express leukocyte common antigen. Green-fluorescent-protein-positive cells were rarely or less frequently found in Ki-67-positive proliferating cells, cytokeratin-positive epithelial cells, or CD31-positive endothelial cells. BMDCs frequently transdifferentiated into subepithelial myofibroblasts and fibroblasts, and often continued to reside in the colonic subepithelia after the experimental colitis had healed. In conclusion, our data indicate the fate of BMDCs, which might be involved in the healing process of the colon after DSS-induced colitis. Our data show that BMDCs contribute to colonic interstitial cells after the colitis has healed. Understanding the fate of BMDCs may be important for stem cell therapy by BMDCs.

Geranylgeranylacetone induces cyclooxygenase-2 expression in cultured rat gastric epithelial cells through NF-kappaB.

Geranylgeranylacetone (GGA) effectively protects the gastric mucosa against noxious agents. The precise mechanisms underlying the gastroprotective actions of GGA are not known. To elucidate the precise mechanism of GGA, the effect of GGA treatment on COX-2 expression in rat gastric epithelial (RGM1) cells was investigated. We used a prostaglandin E2 (PGE2) enzyme-linked immunoassay kit and Western blot analysis to measure PGE2 production and COX-2 induction by GGA treatment in serum-starved RGM1 cells. Gel-shift assay, Western blot analysis, and a reporter assay were performed to determine which COX-2 promoter was involved in GGA-induced COX-2 expression. GGA treatment dose dependently increased COX-2 expression and PGE2 production. The nuclear factor (NF)-kappaB sites of the COX-2 gene promoter were critical for GGA-mediated COX-2 expression. GGA induces COX-2 expression and increases PGE2 production in serum-starved RGM1 cells via activation of the NF-kappaB sites of COX-2 gene promoters.

Novel interleukin-4 and interleukin-1 receptor antagonist gene variations associated with non-cardia gastric cancer in Japan: comprehensive analysis of 207 polymorphisms of 11 cytokine genes.

BACKGROUND AND AIM: Helicobacter pylori (H. pylori)-induced chronic atrophic gastritis is a high-risk factor for gastric cancer. Immune responses to H. pylori are involved in gastric mucosal inflammation, and might affect clinical outcome, including the development of gastric cancer. The present study examines the significance of gene polymorphisms of various cytokines in the development of gastric cancer following H. pylori infection. METHODS: One hundred Japanese non-cardia gastric cancer patients and 93 dyspeptic patients as controls were enrolled in the study (age range 50-75 years). All patients were positive for H. pylori. Genomic DNA was extracted from peripheral whole blood leukocytes, and we comprehensively analyzed 207 single nucleotide polymorphisms (SNP) in 11 cytokine genes; interleukin (IL)-1alpha, IL-1beta, IL-1 receptor antagonist (RN), IL-4, IL-4R, IL-8, IL-10, IL-12, TNF-alpha, TNF-beta, and IFN-gamma, using either invader assay (163 SNP), direct sequencing (22 SNP), or PCR-restriction fragment length polymorphism (22 SNP). RESULTS: Among the 207 SNP examined, the IL-4 gene diplotypes (984 and 2983 AA/GA) had a significant negative association with gastric cancer development (odds ratio =0.3, 95% confidence interval =0.1-0.9). When we adopted the dyspeptic patients over 66 years of age as the controls, the IL-1RN gene diplotypes (-1102 and 6110 CG/GA) also had a significant negative association (odds ratio =0.2, 95% confidence interval =0.1-0.7). CONCLUSION: A comprehensive analysis of 207 SNP of 11 cytokine genes revealed that variations in IL-4 and IL-1RN genes are negatively associated with the risk of developing gastric cancer following H. pylori infection. Distinct host cytokine responses in the gastric mucosa might have a role in H. pylori-induced carcinogenesis.

Synergistic antitumor effects of celecoxib with 5-fluorouracil depend on IFN-gamma.

Cyclooxygenase-2 (COX-2) inhibitors are effective chemopreventive agents against colorectal cancers. For treatment of advanced cancers, combination of COX-2 inhibitors and chemotherapy has been attempted, but the results are still controversial. In the present study, the effects of the COX-2 inhibitor celecoxib on the anticancer potential of chemotherapy, and its mechanisms of action were investigated in point of the angiogenesis, using an advanced cancer model in mice. BALB/c mice were inoculated with colon 26 cells. After the allograft grew up to 5 mm in diameter, the animals received celecoxib, 5FU, or a combination of 5FU and celecoxib (5FU/celecoxib). After 21-days of the treatment, 5FU/celecoxib significantly inhibited the tumor growth and the tumor vessel density compared with the other groups. Celecoxib, 5FU or 5FU/celecoxib significantly suppressed the VEGF content in tumor tissues. 5FU/celecoxib also enhanced IFN-gamma levels in tumor tissue, which could be involved in the potent antitumor effects of 5FU/celecoxib. This hypothesis was proven, because in IFN-gamma knockout (IFN-gamma(-/-)) mice, the inhibitory effects of 5FU/celecoxib on angiogenesis and tumor growth were significantly impaired compared with that in wild type mice. These results suggest that celecoxib enhances the antitumor effect of 5FU against an advanced colon cancer model by suppressing angiogenesis. In addition to VEGF, IFN-gamma has pivotal roles in tumor suppression induced by celecoxib.

East Asian-type Helicobacter pylori cytotoxin-associated gene A protein has a more significant effect on growth of rat gastric mucosal cells than the Western type.

BACKGROUND AND AIM: Cytotoxin-associated gene A (CagA) protein from H. pylori was reported to be injected into host gastric epithelial cells via a bacterial type IV secretion system, thereby modifying signal transduction. It is classified into two major subtypes, Western and East Asian. The present study aimed to compare the effects of East Asian-type and Western-type CagA on host cell growth. METHODS: A tetracycline (tet)-off system and cagA genes from Western and East Asian-type H. pylori (NCTC 11637 and F32) were transfected into untransformed rat gastric mucosal (RGM1) cells to establish RGM1-CagA cell lines in which CagA expression could be controlled by tetracycline. These cell lines were used to investigate the effect of CagA protein expression on cell growth with BrdU and water-soluble tetrazolium reagent (WST-8) assays. CagA expression, phosphorylation and extracellular signal-regulated kinase (ERK) activation were examined with immunoprecipitation and Western blotting analysis. RESULTS: 5-Bromo-2'deoxyuridine (BrdU) and WST-8 assays demonstrated significant increases in DNA replication and RGM1 cell growth after CagA protein expression. ERK phosphorylation was enhanced when CagA protein was expressed in RGM1-CagA cells. Moreover, the East Asian-type CagA showed a significantly greater effect on ERK activation and host cell growth than the Western type. PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor, suppressed ERK phosphorylation and the CagA protein-induced increase in RGM1-CagA cell growth. CONCLUSIONS: CagA protein expression induces an increase in RGM1-CagA cell proliferation via the mitogen-activated protein kinase (MAPK) signal pathway. The East Asian-type CagA showed a significantly greater effect on cell growth than the Western type, suggesting that the East Asian CagA-positive strain may have an important role in pathogenesis.

Helicobacter pylori infection affects Toll-like receptor 4 expression in human gastric mucosa.

BACKGROUND/AIMS: Toll-like receptor 4 (TLR4), which requires a helper molecule, MD-2, is a main receptor for lipopolysaccharide (LPS) from gram-negative bacteria. The expression of TLR4 in H. pylori infection in human gastric mucosa, however, is unclear. The aim of this study was to determine the effect of H. pylori infection on the TLR4 and MD-2 expression in human gastric mucosa. METHODOLOGY: Biopsy samples from the antrum and corpus were obtained from 45 patients (25 patients without H. pylori infection including 5 patients with successful eradication of H. pylori, and 20 patients with H. pylori infection). These samples were used for TLR4, MD-2 mRNA expression study and immunohistochemical study. RESULTS: In patients without H. pylori infection, the expressions of TLR4 and MD-2 were bigger in the corpus mucosa than in the antral mucosa. In patients with H. pylori infection, the expressions of TLR4 and MD-2 significantly increased in the antral and corpus mucosa. Immunohistochemical study revealed similar results as the TLR4 mRNA expression. After the eradication of H. pylori, the expressions of TLR4 and MD-2 were the same levels in both sites as those in patients without H. pylori infection. CONCLUSIONS: The results indicated that H. pylori infection significantly increased TLR4 and MD-2 expressions in the antral and corpus mucosa.

Influences of Helicobacter pylori on cyclooxygenase-2 expression and prostaglandinE2 synthesis in rat gastric epithelial cells in vitro.

BACKGROUND AND AIM: It is known that cyclooxygenase (COX)-2 is over expressed in gastrointestinal neoplasia and Helicobacter pylori (H. pylori) infection is causally linked to gastric cancer. The present study aimed to elucidate the effects of H. pylori on COX-2 expression and prostaglandinE(2) (PGE(2)) production in a gastric epithelial cell line derived from normal rat gastric mucosa (RGM1). METHOD: H. pylori water extracts were prepared from a supernatant of the H. pylori suspension in distilled water. RGM1 cells were cultured with H. pylori water extracts at the final concentration of 2.5, 5, 10 microg/mL for 24 h. For the time sequence study, RGM1 cells were cultured with 10 microg/mL H. pylori water extracts for 0, 6, 12, 24 and 48 h. COX-1 and COX-2 expression in the RGM1 cells was analyzed by western blotting. The levels of PGE(2) in the cultured media were measured by enzyme immunoassay. RESULTS: H. pylori did not affect COX-1 expression; whereas COX-2 expression increased by six-fold at 24 h after incubation of RGM1 cells with 10 microg/mL H. pylori water extracts. The increase in COX-2 expression was evident after 12 h of incubation; reached a peak at 24 h and declined at 48 h. H. pylori dose dependently increased COX-2 expression and PGE(2) synthesis in RGM1 cells. CONCLUSION: H. pylori induces COX-2 expression and increases PGE(2) synthesis in RGM1 cells in vitro. These results indicate that H. pylori-associated gastric carcinogenesis may depend on COX-2 expression.

Helicobacter pylori eradication to prevent gastric cancer: underlying molecular and cellular mechanisms.

Numerous cellular and molecular events have been described in development of gastric cancer. In this article, we overviewed roles of Helicobacter pylori (H pylori) infection on some of the important events in gastric carcinogenesis and discussed whether these cellular and molecular events are reversible after cure of the infection. There are several bacterial components affecting gastric epithelial kinetics and promotion of gastric carcinogenesis. The bacterium also increases risks of genetic instability and mutations due to NO and other reactive oxygen species. Epigenetic silencing of tumor suppressor genes such as RUNX3 may alter the frequency of phenotype change of gastric glands to those with intestinal metaplasia. Host factors such as increased expression of growth factors, cytokines and COX-2 have been also reported in non-cancerous tissue in H pylori-positive subjects. It is noteworthy that most of the above phenomena are reversed after the cure of the infection. However, some of them including overexpression of COX-2 continue to exist and may increase risks for carcinogenesis in metaplastic or dysplastic mucosa even after successful H pylori eradication. Thus, H pylori eradication may not completely abolish the risk for gastric carcinogenesis. Efficiency of the cure of the infection in suppressing gastric cancer depends on the timing and the target population, and warrant further investigation.

Palmitoylation of LAT contributes to its subcellular localization and stability.

Palmitoylation is a protein modification for trafficking to lipid raft. Without palmitoylation, linker for activation of T cells (LAT), an adaptor molecule mediating T cell receptor signaling, is unable to localize in lipid rafts and to mediate T cell activation. We here show a novel role for palmitoylation in LAT trafficking to the plasma membrane and in the stability of the LAT protein. The human LAT mutant lacking palmitoylation was unable to traffic to the plasma membrane despite the presence of transmembrane portion. The mouse LAT mutant lacking palmitoylation was unstable and susceptible to degradation via the proteasome pathway. The human LAT mutant became unstable when the extracellular portion was swapped for that from mouse, indicating that both palmitoylation and the extracellular portion regulate the stability of LAT. These results suggest that palmitoylation has an important role in trafficking to the plasma membrane and the stability of LAT.

Oral glucose tolerance test predicts prognosis of patients with liver cirrhosis.

OBJECTIVE: The aim of this study was to evaluate whether oral glucose tolerance test (OGTT) was useful in evaluating the prognosis of patients with liver cirrhosis. METHODS: Fifty-six patients with liver cirrhosis were enrolled in a prospective cohort study. In all cases, glucose tolerance was diagnosed by a 75-g OGTT according to World Health Organization (WHO) criteria. The relationship of clinical variables to the cirrhosis-related prognosis was investigated using univariate and multivariate regression models. RESULTS: Diabetes mellitus (DM) was diagnosed in 21 subjects (38%), impaired glucose tolerance (IGT) in 13 subjects (23%), and normal glucose tolerance (NGT) in 22 subjects (39%) using OGTT. The cumulative survival rates of patients with liver cirrhosis and NGT were 94.7% at 5 yr; liver cirrhosis and IGT, 68.8% at 5 yr; liver cirrhosis and DM, 56.6% at 5 yr. The survival rates of patients with liver cirrhosis and DM significantly differed from those with NGT. Univariate analysis demonstrated that serum albumin, total bilirubin, prothrombin activity, Child-Pugh scores, and glucose intolerance were highly significant prognostic factors. Multiple regression analysis yielded albumin and DM as the most powerful independent negative predictors of survival. CONCLUSIONS: OGTT appears to be useful for evaluating the prognosis of cirrhotic patients.

Endothelin-1, an ulcer inducer, promotes gastric ulcer healing via mobilizing gastric myofibroblasts and stimulates production of stroma-derived factors.

Endothelin (ET)-1 is a potent inducer of peptic ulcers. The roles of ET-1 in ulcer healing, however, have remained unclear, and these were investigated in mice. Gastric ulcers were induced in mice by serosal application of acetic acid. Three days later, mice were given a neutralizing ET-1 antibody or nonimmunized serum. The ulcer size, amount of fibrosis and myofibroblasts, and localization of ET-1 and ET(A/B) receptors were analyzed. To elucidate the mechanisms underlying the effects of ET-1, we examined the proliferation, migration, and release of growth and angiogenic factors in gastric myofibroblasts with or without ET-1. The expression of prepro-ET-1 (an ET-1 precursor) and ET-converting enzyme-1 was examined in gastric myofibroblasts using RT-PCR. Immunoneutralization of ET-1 delayed gastric ulcer healing. The areas of fibrosis and myofibroblasts were smaller in the anti-ET-1 antibody group than in the control. ET-1 was expressed in the gastric epithelium, myofibroblasts, and other cell types. ET(A) receptors, but not ET(B) receptors, were present in myofibroblasts. ET-1 increased proliferation and migration of gastric myofibroblasts. ET-1 stimulated the release of hepatocyte growth factor, VEGF, PGE(2), and IL-6 from gastric myofibroblasts. mRNA for prepro-ET-1 and ET-converting enzyme-1 was also expressed. ET-1 promotes the accumulation of gastric myofibroblasts and collagen fibrils at gastric ulcers. ET-1 also stimulates migration and proliferation of gastric myofibroblasts and enhances the release of growth factors, angiogenic factors, and PGE(2). Thus ET-1 has important roles not only in ulcer formation but also in ulcer healing via mobilizing myofibroblasts and inducing production of stroma-derived factors.

Fucosylated haptoglobin is a novel marker for pancreatic cancer: a detailed analysis of the oligosaccharide structure and a possible mechanism for fucosylation.

Changes in oligosaccharide structures have been reported in certain types of malignant transformations and, thus, could be used for tumor markers in certain types of cancer. In the case of pancreatic cancer cell lines, a variety of fucosylated proteins are secreted into their conditioned media. To identify fucosylated proteins in the serum of patients with pancreatic cancer, we performed western blot analyses using Aleuria Aurantica Lectin (AAL), which is specific for fucosylated structures. An approximately 40 kD protein was found to be highly fucosylated in pancreatic cancer and an N-terminal analysis revealed that it was the beta chain of haptoglobin. While the appearance of fucosylated haptoglobin has been reported in other diseases such as hepatocellular carcinoma, liver cirrhosis, gastric cancer and colon cancer, the incidence was significantly higher in the case of pancreatic cancer. Fucosylated haptoglobin was observed more frequently at the advanced stage of pancreatic cancer and disappeared after an operation. A mass spectrometry analysis of haptoglobin purified from the serum of patients with pancreatic cancer and the medium from a pancreatic cancer cell line, PSN-1, showed that the alpha 1-3/alpha 1-4/alpha 1-6 fucosylation of haptoglobin was increased in pancreatic cancer. When a hepatoma cell line, Hep3B, was cultured with the conditioned media from pancreatic cancer cells, haptoglobin secretion was dramatically increased. These findings suggest that fucosylated haptoglobin could serve as a novel marker for pancreatic cancer. Two possibilities were considered in terms of the fucosylation of haptoglobin. One is that pancreatic cancer cells, themselves, produce fucosylated haptoglobin; the other is that pancreatic cancer produces a factor, which induces the production of fucosylated haptoglobin in the liver.